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91.
Nobukatsu Horita Kiichiro Tsuchiya Ryohei Hayashi Keita Fukushima Shuji Hibiya Masayoshi Fukuda Yoshihito Kano Tomohiro Mizutani Yasuhiro Nemoto Shiro Yui Ryuichi Okamoto Tetsuya Nakamura Mamoru Watanabe 《Biochemical and biophysical research communications》2014
Background and aims
The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.Methods
Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.Results
We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.Conclusions
The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus. 相似文献92.
Takaharu Yamamoto Junko Mochida Jun Kadota Miyoko Takeda Erfei Bi Kazuma Tanaka 《Molecular biology of the cell》2010,21(7):1237-1252
The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast. 相似文献
93.
Atsuzawa K Nakazawa A Mizutani K Fukasawa M Yamamoto N Hashimoto T Usuda N 《Histochemistry and cell biology》2010,134(6):565-579
The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids
are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by
glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult
rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation
enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis
for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount
of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron
microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal
neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation
enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously. 相似文献
94.
Yasushi Todoroki Kumi Naiki Hikaru Aoyama Minaho Shirakura Kotomi Ueno Masaharu Mizutani Nobuhiro Hirai 《Bioorganic & medicinal chemistry letters》2010,20(18):5506-5509
The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8′-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp2 nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect. 相似文献
95.
Yosuke Ichijima Ken-ichi Yoshioka Yoshiko Yoshioka Keitaro Shinohe Hiroaki Fujimori Junya Unno Masatoshi Takagi Hidemasa Goto Masaki Inagaki Shuki Mizutani Hirobumi Teraoka 《PloS one》2010,5(1)
During tumorigenesis, cells acquire immortality in association with the
development of genomic instability. However, it is still elusive how genomic
instability spontaneously generates during the process of tumorigenesis. Here,
we show that precancerous DNA lesions induced by oncogene acceleration, which
induce situations identical to the initial stages of cancer development, trigger
tetraploidy/aneuploidy generation in association with mitotic aberration.
Although oncogene acceleration primarily induces DNA replication stress and the
resulting lesions in the S phase, these lesions are carried over into the M
phase and cause cytokinesis failure and genomic instability. Unlike directly
induced DNA double-strand breaks, DNA replication stress-associated lesions are
cryptogenic and pass through cell-cycle checkpoints due to limited and
ineffective activation of checkpoint factors. Furthermore, since damaged M-phase
cells still progress in mitotic steps, these cells result in chromosomal
mis-segregation, cytokinesis failure and the resulting tetraploidy generation.
Thus, our results reveal a process of genomic instability generation triggered
by precancerous DNA replication stress. 相似文献
96.
The relationship between the allelopathic p-menthane-3,8-diols and the ontogenetic age in Eucalyptus citriodora was elucidated. The diols in the soil from a Eucalyptus grove were analysed by mass chromatography. Germination and growth inhibitory activities of the cis-diol against several higher plants were examined. 相似文献
97.
Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate 总被引:3,自引:0,他引:3
Sphingolipids are major lipid constituents of the eukaryotic plasma membrane. Without certain sphingolipids, cells and/or embryos cannot survive, indicating that sphingolipids possess important physiological functions that are not substituted for by other lipids. One such role may be signaling. Recent studies have revealed that some sphingolipid metabolites, such as long-chain bases (LCBs; sphingosine (Sph) in mammals), long-chain base 1-phosphates (LCBPs; sphingosine 1-phosphate (S1P) in mammals), ceramide (Cer), and ceramide 1-phosphate (C1P), act as signaling molecules. The addition of phosphate groups to LCB/Sph and Cer generates LCBP/S1P and C1P, respectively. These phospholipids exhibit completely different functions than those of their precursors. In this review, we describe recent advances in understanding the functions of LCBP/S1P and C1P in mammals and in the yeast Saccharomyces cerevisiae. Since LCB/Sph, LCBP/S1P, Cer, and C1P are mutually convertible, regulation of not only the total amount of the each lipid but also of the overall balance in cellular levels is important. Therefore, we describe in detail their metabolic pathways, as well as the genes involved in each reaction. 相似文献
98.
Kanno T Kawanishi M Takamura-Enya T Arlt VM Phillips DH Yagi T 《Mutation research》2007,634(1-2):184-191
3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a (32)P-postlabeling/thin layer chromatography (TLC) method and a (32)P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 microM) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N(6)-C2-ABA) and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N(2)-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3'p-N(6)-C2-ABA and its relative adduct labeling (RAL) value at 36.4 microM of 3-NBA was 200.8+/-86.1/10(8)nucleotide. 相似文献
99.
CC chemokine receptor 7 contributes to Gi-dependent T cell motility in the lymph node 总被引:10,自引:0,他引:10
Naive T cells migrate extensively within lymph node (LN) T zones to scan for Ag-bearing dendritic cells. However, the extracellular signals controlling T cell motility in LNs are not well defined. In this study, by real-time imaging of LNs, we show that the inhibition of Gi signaling in T cells severely impairs their migration. The chemokine CCL21, a ligand of CCR7, strongly induces chemokinesis in vitro, and T cell motility in LNs from CCR7 ligand-deficient plt/plt mice was reduced. CCR7-deficient T cells in wild-type LNs showed a similar reduction in motility, and antagonism of CXCR4 function did not further decrease their motility. The effect of CCR7 or CCR7-ligand deficiency could account for approximately 40% of the Gi-dependent motility. These results reveal a role for CCR7 in promoting T cell migration within lymphoid organ T zones, and they suggest the additional involvement of novel Gi-coupled receptors in promoting T cell motility at these sites. 相似文献
100.
Amino acid substitutions in the s2 region enhance severe acute respiratory syndrome coronavirus infectivity in rat angiotensin-converting enzyme 2-expressing cells
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Fukushi S Mizutani T Sakai K Saijo M Taguchi F Yokoyama M Kurane I Morikawa S 《Journal of virology》2007,81(19):10831-10834
To clarify the molecular basis of severe acute respiratory syndrome coronavirus (SARS-CoV) adaptation to different host species, we serially passaged SARS-CoV in rat angiotensin-converting enzyme 2 (ACE2)-expressing cells. After 15 passages, the virus (Rat-P15) came to replicate effectively in rat ACE2-expressing cells. Two amino acid substitutions in the S2 region were found on the Rat-P15 S gene. Analyses of the infectivity of the pseudotype-bearing S protein indicated that the two substitutions in the S2 region, especially the S950F substitution, were responsible for efficient infection. Therefore, virus adaptation to different host species can be induced by amino acid substitutions in the S2 region. 相似文献